Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast

Inada, Maki; Nichols, Robert J.; Parsa, Jahan Yar; Homer, Christina M.; Benn, Ruby A.; Hoxie, Reyal S.; Madhani, Hiten D.; Shuman, Stewart; Schwer, Beate; Pleiss, Jeffrey A.

doi:10.1093/nar/gkw603

Cited by 9 publications

(12 citation statements)

References 36 publications

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“…ChIP-seq was conducted as previously described 67 . ChIP-Seq immunoprecipitations were performed with 10 µg anti-H3K9me2 (Abcam, #ab1220), and 10 µg anti-FLAG (Sigma, #P3165).…”

Section: Chip-seqmentioning

confidence: 99%

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Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly

Parsa

Boudoukha

Burke

et al. 2017

Preprint

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peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 3 To understand how Seb1 interfaces with the transcription of dg and dh repeats to promote heterochromatin, we employed a previously identified viable, heterochromatindefective allele, seb1-1 15 . When combined with mutants in the RNAi machinery, seb1-1 eliminates pericentromeric heterochromatin, while the corresponding single mutants decrease H3K9me, indicative of partially redundant pathways 15 . We examined transcription of heterochromatin at single-nucleotide resolution and tested the impact of the seb1-1 allele using Nascent Elongating Transcript Sequencing (NET-Seq) 20 . To analyze the intrinsic transcriptional properties of heterochromatic sequences prior to the establishment of heterochromatin assembly, we used the clr4∆ mutant, which lacksH3K9me and displays full derepression of most silenced chromatin regions. We compared this strain to a clr4∆ seb1-1 double mutant to assess the impact of seb1-1.We first examined the effect of seb1-1 on transcription of non-heterochromatic regions peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 4 Extended Data Figure 1). The seb1-1 mutation causes a reduced median 5' traveling ratio and an increased median 3' traveling ratio for both clusters ( for p values). These data indicate that the seb1-1 allele leads to decreased RNAPII pausing at gene 5' ends with an associated increased 3' signal; the latter may be due to polymerase release from upstream pauses. peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 5 To compare the binding of Seb1 across transcript classes, we computed the fraction of RNA covered by Seb1 PAR-CLIP read clusters. We observed a ~12-fold higher PAR-CLIP cluster coverage for pericentromeric repeat intervals than for coding gene intervals (Figure 2d and Extended Data Figure 4a). Non-coding RNAs display the highest coverage at a mean level ~100-fold higher than that of coding-genes (Extended Data Figure 4b).We next examined the NET-Seq profiles of pericentromeric heterochromatin sequences of clr4∆ and clr4∆ seb1-1 strains (replicate experiments were conducted). The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 6 Figure 4d).These data reveal detectable Seb1-dependent RNAPII pauses in pericentromeric sequences. (Figure 2g). Silencing of ura4 + was determined using YS-FOA plates, which selects for ura4+ repression. The insert of Fragment ...

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“…ChIP-seq was conducted as previously described 67 . ChIP-Seq immunoprecipitations were performed with 10 µg anti-H3K9me2 (Abcam, #ab1220), and 10 µg anti-FLAG (Sigma, #P3165).…”

Section: Chip-seqmentioning

confidence: 99%

“…ChIP-seq analysis was conducted as previously described 67 . Briefly, adaptor sequences from ChIP-seq sequencing libraries were removed and reads <20nt were omitted.…”

Section: Chip-seq Analysismentioning

confidence: 99%

Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly

Parsa

Boudoukha

Burke

et al. 2017

Preprint

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“…Following overnight incubation at 65°C, ChIP and Input samples were purified using Machery Nagel PCR clean up kit. Library preparation for sequencing was performed as described [72,73]. Samples were sequenced on a HiSeq 4000 platform (Illumina) with a Single End 50 run.…”

Section: Chip-seq Sample and Library Preparationmentioning

confidence: 99%

Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

Greenstein

Barrales

Sanchez

et al. 2019

Preprint

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Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While hetero-and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary, using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Interestingly, this effect can be gene orientation dependent. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. At least two independent pathways direct this Set1 activity-inhibition of catalysis by Suv39/Clr4 and disruption of nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

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“…ChIP-seq was conducted as described previously (Inada et al 2016). ChIP-seq immunoprecipitations were performed with 10 μg of anti-H3K9me2 (Abcam, ab1220) and 10 µg of anti-Flag (Sigma, P3165).…”

Section: Chip-seqmentioning

confidence: 99%

“…ChIP-seq analysis was conducted as described previously (Inada et al 2016). Briefly, adaptor sequences from ChIP-seq sequencing libraries were removed (using GATCGGAAGA), and reads <20 nt were omitted.…”

Section: Chip-seq Analysismentioning

confidence: 99%

Polymerase pausing induced by sequence-specific RNA-binding protein drives heterochromatin assembly

et al. 2018

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In , transcripts derived from the pericentromeric and repeats promote heterochromatin formation via RNAi as well as an RNAi-independent mechanism involving the RNA polymerase II (RNAPII)-associated RNA-binding protein Seb1 and RNA processing activities. We show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and that their presence correlates with the heterochromatin-triggering activities of the corresponding and DNA fragments. Globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking RNAi. These results uncover Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.

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Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast

Cited by 9 publications

References 36 publications

Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly

Polymerase pausing induced by sequence-specific RNA binding protein drives heterochromatin assembly

Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability

Polymerase pausing induced by sequence-specific RNA-binding protein drives heterochromatin assembly

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